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Thioredoxins function as deglutathionylase enzymes in the yeast Saccharomyces cerevisiae.

Greetham D, Vickerstaff J, Shenton D, Perrone GG, Dawes IW, Grant CM - BMC Biochem. (2010)

Bottom Line: Glutaredoxins and thioredoxins are key oxidoreductases which have been implicated in regulating glutathionylation/deglutathionylation in diverse organisms.We have confirmed that thioredoxins, but not glutaredoxins, catalyse deglutathionylation of model glutathionylated substrates using purified thioredoxin and glutaredoxin proteins.Furthermore, we show that the deglutathionylase activity of thioredoxins is required to reduce the high levels of glutathionylation in stationary phase cells, which occurs as cells exit stationary phase and resume vegetative growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: The University of Manchester, Faculty of Life Sciences, Manchester M13 9PT, UK.

ABSTRACT

Background: Protein-SH groups are amongst the most easily oxidized residues in proteins, but irreversible oxidation can be prevented by protein glutathionylation, in which protein-SH groups form mixed disulphides with glutathione. Glutaredoxins and thioredoxins are key oxidoreductases which have been implicated in regulating glutathionylation/deglutathionylation in diverse organisms. Glutaredoxins have been proposed to be the predominant deglutathionylase enzymes in many plant and mammalian species, whereas, thioredoxins have generally been thought to be relatively inefficient in deglutathionylation.

Results: We show here that the levels of glutathionylated proteins in yeast are regulated in parallel with the growth cycle, and are maximal during stationary phase growth. This increase in glutathionylation is not a response to increased reactive oxygen species generated from the shift to respiratory metabolism, but appears to be a general response to starvation conditions. Our data indicate that glutathionylation levels are constitutively high in all growth phases in thioredoxin mutants and are unaffected in glutaredoxin mutants. We have confirmed that thioredoxins, but not glutaredoxins, catalyse deglutathionylation of model glutathionylated substrates using purified thioredoxin and glutaredoxin proteins. Furthermore, we show that the deglutathionylase activity of thioredoxins is required to reduce the high levels of glutathionylation in stationary phase cells, which occurs as cells exit stationary phase and resume vegetative growth.

Conclusions: There is increasing evidence that the thioredoxin and glutathione redox systems have overlapping functions and these present data indicate that the thioredoxin system plays a key role in regulating the modification of proteins by the glutathione system.

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Modification of proteins by glutathionylation. This reversible post-translational modification involves the formation of a mixed disulphide between a free thiol group on a protein and a molecule of glutathione. This may occur through oxidation of a protein-thiol group in response to ROS, and reaction with GSH as shown in the diagram. Alternatively, oxidized GSSG may react with protein-SH groups (for a review, see [9]). Deglutathionylation may be catalysed by glutaredoxin (Grx) or thioredoxin (Trx).
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Figure 1: Modification of proteins by glutathionylation. This reversible post-translational modification involves the formation of a mixed disulphide between a free thiol group on a protein and a molecule of glutathione. This may occur through oxidation of a protein-thiol group in response to ROS, and reaction with GSH as shown in the diagram. Alternatively, oxidized GSSG may react with protein-SH groups (for a review, see [9]). Deglutathionylation may be catalysed by glutaredoxin (Grx) or thioredoxin (Trx).

Mentions: Glutathionylation is the major form of S-thiolation in eukaryotic cells. This reversible post-translational modification involves the formation of a mixed disulphide between a free thiol on a protein and a molecule of glutathione (GSH) (Fig. 1). It is particularly important since it can both protect cysteine residues from irreversible oxidation and can also regulate the activity of many target proteins. Greater than 150 targets of modification have been identified from eukaryotic species affecting diverse processes including glycolysis, protein synthesis, protein degradation, signal transduction and transport [3,4]. In many cases, this protein modification is implicated in the regulation of protein function and activity; examples include the HIV-1 protease [5], ubiquitin-conjugating enzymes in bovine retina cells [6], DNA binding by the transcription factor c-Jun [7] and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [8]. Protein glutathionylation is a dynamic process that occurs in cells under physiological conditions, as well as following exposure to an oxidative stress. Models have been proposed in which this protein modification does not require an enzymatic activity, but proceeds via the reaction of partially oxidised protein sulphydryls with GSH, or by thiol/disulphide exchange reactions with the oxidised disulphide form of glutathione (Fig. 1) [9]. There does not appear to be any unifying feature of target proteins, and the fact that not all -SH containing proteins are modified in response to an oxidative stress suggests that this protein modification must be tightly regulated [10,11].


Thioredoxins function as deglutathionylase enzymes in the yeast Saccharomyces cerevisiae.

Greetham D, Vickerstaff J, Shenton D, Perrone GG, Dawes IW, Grant CM - BMC Biochem. (2010)

Modification of proteins by glutathionylation. This reversible post-translational modification involves the formation of a mixed disulphide between a free thiol group on a protein and a molecule of glutathione. This may occur through oxidation of a protein-thiol group in response to ROS, and reaction with GSH as shown in the diagram. Alternatively, oxidized GSSG may react with protein-SH groups (for a review, see [9]). Deglutathionylation may be catalysed by glutaredoxin (Grx) or thioredoxin (Trx).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2836980&req=5

Figure 1: Modification of proteins by glutathionylation. This reversible post-translational modification involves the formation of a mixed disulphide between a free thiol group on a protein and a molecule of glutathione. This may occur through oxidation of a protein-thiol group in response to ROS, and reaction with GSH as shown in the diagram. Alternatively, oxidized GSSG may react with protein-SH groups (for a review, see [9]). Deglutathionylation may be catalysed by glutaredoxin (Grx) or thioredoxin (Trx).
Mentions: Glutathionylation is the major form of S-thiolation in eukaryotic cells. This reversible post-translational modification involves the formation of a mixed disulphide between a free thiol on a protein and a molecule of glutathione (GSH) (Fig. 1). It is particularly important since it can both protect cysteine residues from irreversible oxidation and can also regulate the activity of many target proteins. Greater than 150 targets of modification have been identified from eukaryotic species affecting diverse processes including glycolysis, protein synthesis, protein degradation, signal transduction and transport [3,4]. In many cases, this protein modification is implicated in the regulation of protein function and activity; examples include the HIV-1 protease [5], ubiquitin-conjugating enzymes in bovine retina cells [6], DNA binding by the transcription factor c-Jun [7] and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [8]. Protein glutathionylation is a dynamic process that occurs in cells under physiological conditions, as well as following exposure to an oxidative stress. Models have been proposed in which this protein modification does not require an enzymatic activity, but proceeds via the reaction of partially oxidised protein sulphydryls with GSH, or by thiol/disulphide exchange reactions with the oxidised disulphide form of glutathione (Fig. 1) [9]. There does not appear to be any unifying feature of target proteins, and the fact that not all -SH containing proteins are modified in response to an oxidative stress suggests that this protein modification must be tightly regulated [10,11].

Bottom Line: Glutaredoxins and thioredoxins are key oxidoreductases which have been implicated in regulating glutathionylation/deglutathionylation in diverse organisms.We have confirmed that thioredoxins, but not glutaredoxins, catalyse deglutathionylation of model glutathionylated substrates using purified thioredoxin and glutaredoxin proteins.Furthermore, we show that the deglutathionylase activity of thioredoxins is required to reduce the high levels of glutathionylation in stationary phase cells, which occurs as cells exit stationary phase and resume vegetative growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: The University of Manchester, Faculty of Life Sciences, Manchester M13 9PT, UK.

ABSTRACT

Background: Protein-SH groups are amongst the most easily oxidized residues in proteins, but irreversible oxidation can be prevented by protein glutathionylation, in which protein-SH groups form mixed disulphides with glutathione. Glutaredoxins and thioredoxins are key oxidoreductases which have been implicated in regulating glutathionylation/deglutathionylation in diverse organisms. Glutaredoxins have been proposed to be the predominant deglutathionylase enzymes in many plant and mammalian species, whereas, thioredoxins have generally been thought to be relatively inefficient in deglutathionylation.

Results: We show here that the levels of glutathionylated proteins in yeast are regulated in parallel with the growth cycle, and are maximal during stationary phase growth. This increase in glutathionylation is not a response to increased reactive oxygen species generated from the shift to respiratory metabolism, but appears to be a general response to starvation conditions. Our data indicate that glutathionylation levels are constitutively high in all growth phases in thioredoxin mutants and are unaffected in glutaredoxin mutants. We have confirmed that thioredoxins, but not glutaredoxins, catalyse deglutathionylation of model glutathionylated substrates using purified thioredoxin and glutaredoxin proteins. Furthermore, we show that the deglutathionylase activity of thioredoxins is required to reduce the high levels of glutathionylation in stationary phase cells, which occurs as cells exit stationary phase and resume vegetative growth.

Conclusions: There is increasing evidence that the thioredoxin and glutathione redox systems have overlapping functions and these present data indicate that the thioredoxin system plays a key role in regulating the modification of proteins by the glutathione system.

Show MeSH
Related in: MedlinePlus